Mitochondria are essential organelles found in most eukaryotic cells and provide for diverse metabolic functions including the production of energy and specialized metabolites. Mitochondria require proteins and RNAs coded both by the mitochondrial and nuclear genomes. In humans, at least 1000 nuclear encoded proteins are imported into mitochondria, whereas 13 proteins are coded by mitochondrial genes. The translation of this limited number of mitochondrial coded proteins requires mitochondrial ribosomes, translation factors, and transfer RNAs. Recent studies in evolutionarily diverse organisms have shown that various RNA components are coded in the nucleus and imported from the cytosol into mitochondria. Whereas the understanding of import pathways for mitochondrial proteins is very detailed, very little is understood about the mechanism of RNA import, let alone the specific RNA substrates that are imported. The overall goal of this proposal is to dissect the pathway of RNA import into mitochondria, identify the RNA species that are imported, and determine pathological consequences when RNA import is inhibited. The RNA-binding enzyme PNPase localizes to the mitochondrial intermembrane space and seems to function as a receptor for RNAs that are imported from the cytosol. In addition, PNPase is only present in organisms such as flies, worms, and mammals, suggesting it functions in higher eukaryotes. To accomplish the proposed goals, the following specific aims are planned: In Aim 1, the biochemical role of PNPase in the import and biogenesis of RNA in the mitochondrion will be determined. In Aim 2, a conditional mouse model in which PNPase can be knocked down in a controlled manner will be characterized to determine the physiologic function of PNPase and the consequences of PNPase loss in mitochondrial function. Finally, in Specific Aim 3, the role of PNPase in RNA import in vivo will be investigated. A mutation in PNPase has been linked to a neurodegenerative disease, suggesting PNPase plays an important role in health and disease. In addition to the fundamental knowledge, characterization of the RNA import pathway can open a new door to develop methods for treating severe pathological diseases associated with mutations in mitochondrial tRNAs or mitochondrial genes. This application has a broader impact in public health because mutations in PNPase have been linked to neurodegeneration. PUBLIC HEALTH RELEVANCE: This proposal is relevant to public health because mutations in the gene PNPase have been linked to a hereditary neurodegenerative disease. Characterization of this disease will provide insight into how mitochondrial dysfunction contributes to neurodegeneration.